Hepatocyte growth factor (HGF) was found as a protein having hepatocyte proliferation action, and subsequent studies have shown that the HGF is a physiologically active protein having various kinds of pharmacological activities other than the proliferation action of hepatocytes. The pharmacological activities are described, for example, in Jikken Igaku zoukan 10(3) pp. 330-339 (1992).
Based on the diverse activities of HGF, other names of HGF including SF (scatter factor) and TCF (tumor cytotoxic factor) are used. However, in the present invention, these known proteins having proliferation action of hepatocytes are collectively referred to as HGF. Because of its pharmacological activities, expected is the development of HGF as a cirrhosis drug, a renal disease drug, an epithelial cell proliferation accelerant, an anticancer drug, an anti-side-effect drug for cancer therapy, a lung disorder drug, a gastroduodenal damage drug, a cranial nerve disorder drug, an immunosuppressive anti-side-effect drug, a collagen-degrading promoter, a cartilage disorder drug, an arterial disease drug, a pulmonary fibrosis drug, a liver disorder drug, a blood coagulation disorder drug, a hypoproteinemia drug, a wound healing drug, a neuropathy drug, a hematopoietic stem cell augmenter and hair growth promotion drug, etc. (see, for example, JP-A No. 4-18028, JP-A No. 4-49246, JP-A No. 7-179356, JP-A No. 6-25010, JP-A No. 6-340546, JP-A No. 6-172207, JP-A No. 7-89869, JP-A No. 6-40934, JP-A No. 6-503949, JP-A No. 6-40935, JP-A No. 6-56692, JP-A No. 7-41429, JP3395181 and JP-A No. 5-213721).
HGF is secreted from organs such as the liver, kidneys, lungs, brain, bone marrow, spleen, and placenta, or from blood cells such as platelets and leukocytes. However, as the in vivo content of HGF is infinitesimal, in order to use HGF as a medicinal preparation, a large amount of HGF should be produced by a genetic engineering technique using cells. It is conventionally known that HGF can be produced using animal cells such as Chinese hamster ovary (CHO) cells (see, for example, JP-A No. 11-4696 and JP-A No. 10-191991).
In cultivating animal cells, fetal bovine serum has conventionally been added. However, recently, serum-free cultivation has been advanced. Therefore, to produce a protein for use as a medicinal preparation with the use of animal cells such as CHO cells, cultivation is generally performed under a serum-free condition. This is because by not using fetal bovine serum, production costs can be reduced and the risk of contamination of viruses and abnormal prions derived from fetal bovine serum can be avoided. Even when HGF is produced using animal cells such as CHO cells, serum-free cultivation can be employed. In this case, however, there is a problem that HGF can be produced only as an inactive HGF precursor protein.
In the biosynthesis of HGF, single-stranded HGF precursor protein is first synthesized and secreted from cells. This HGF precursor protein is an inactive precursor. It is not until the HGF precursor protein is cleaved by the action of a protease called HGF activator (HGFA) and converted into a double stranded structure that the HGF becomes active. This activated HGF is a heterodimer comprising an α chain and a β chain. HGFA itself is also originally biosynthesized as an inactive single-stranded precursor (hereinafter also referred to as pro-HGFA), and is normally circulating in the form of pro-HGFA in the plasma. In the event of tissue injury, working with blood-clotting system and the like, the pro-HGFA is cleaved by the action of thrombin and becomes an active double-stranded HGFA to activate the HGF precursor protein. As serum is in a state where blood-clotting system has already worked, HGFA exists in its active form in serum. Therefore, when CHO cells into which DNA encoding HGF is introduced are cultivated in the presence of serum, HGF produced in culture medium is in its active form by the action of active HGFA in serum. Meanwhile, when the CHO cells are cultivated under a serum-free condition, because of the absence of HGFA, HGF is produced only as an inactive HGF precursor protein. Although it might be a possible option that HGFA, instead of serum, is added to the cultivation system of CHO cells, there is difficulty in obtaining an active HGFA in the absence of serum because of a cascade that, as described above, HGFA is also secreted as an inactive single-stranded pro-HGFA and then converted into an active HGFA in conjunction with blood-clotting system. Therefore, with conventional technique, an active HGF cannot be efficiently produced unless serum is added.
Consequently, development of a method to activate HGF precursor protein without adding serum has been desired. If such a method becomes available, an active HGF can be safely produced even when CHO cells are cultivated under a serum-free condition, and the risk of contamination of viruses and abnormal prions can be avoided. In addition, serum-free host systems in which yeast and an individual insect, etc. are used as hosts for recombinant production of HGF become available, and the method can be applied to a production system in which HGF expression at a higher level than in CHO cells can be expected.
However, such a method to activate HGF precursor protein without adding serum was previously unknown.